Previously, we showed that the evolution of high acuity vision in fishes was directly associated with their unique pH-sensitive hemoglobins that allow O2 to be delivered to the retina at PO2s more than ten-fold that of arterial blood (Damsgaard et al., 2019). Here, we show strong evidence that vacuolar-type H+-ATPase and plasma-accessible carbonic anhydrase in the vascular structure supplying the retina act together to acidify the red blood cell leading to O2 secretion. In vivo data indicate that this pathway primarily affects the oxygenation of the inner retina involved in signal processing and transduction, and that the evolution of this pathway was tightly associated with the morphological expansion of the inner retina. We conclude that this mechanism for retinal oxygenation played a vital role in the adaptive evolution of vision in teleost fishes.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

The speciose mammalian order Eulipotyphla (moles, shrews, hedgehogs, solenodons) combines an unusual diversity of semi-aquatic, semi-fossorial, and fossorial forms that arose from terrestrial forbearers. However, our understanding of the ecomorphological pathways leading to these lifestyles has been confounded by a fragmentary fossil record, unresolved phylogenetic relationships, and potential morphological convergence, calling for novel approaches. The net surface charge of the oxygen-storing muscle protein myoglobin (ZMb), which can be readily determined from its primary structure, provides an objective target to address this question due to mechanistic linkages with myoglobin concentration. Here, we generate a comprehensive 71 species molecular phylogeny that resolves previously intractable intra-family relationships and then ancestrally reconstruct ZMb evolution to identify ancient lifestyle transitions based on protein sequence alone. Our phylogenetically informed analyses confidently resolve fossorial habits having evolved twice in talpid moles and reveal five independent secondary aquatic transitions in the order housing the world's smallest endothermic divers.

Several parameters are important when choosing the most appropriate animal to model human obstetrics, including gestation period, number of fetuses per gestation and placental structure. The domesticated long-tailed chinchilla (Chinchilla lanigera) is a well-suited and appropriate animal model of pregnancy that often will carry only one offspring and has a long gestation period of 105-115 days. Furthermore, the chinchilla placenta is of the haemomonochorial labyrinthine type and is therefore comparable to the human villous haemomonochorial placenta. This proof-of-concept study demonstrated the feasibility in laboratory settings, and demonstrated the potential of the pregnant chinchilla as an animal model for obstetric research and its potential usefulness for non-invasive measurements in the placenta. We demonstrate measurements of the placental and fetal metabolism (demonstrated in vivo by hyperpolarized MRI and in vitro by qPCR analyses), placental vessels (demonstrated ex vivo by contrast-enhanced CT angiography) and overall anatomy (demonstrated in vivo by whole-body CT).

Comparative anatomy and physiology are disciplines related to structures and mechanisms in three-dimensional (3D) space. For the past centuries, scientific reports in these fields have relied on written descriptions and two-dimensional (2D) illustrations, but in recent years 3D virtual modelling has entered the scene. However, comprehending complex anatomical structures is hampered by reproduction on flat inherently 2D screens. One way to circumvent this problem is in the production of 3D-printed scale models. We have applied computed tomography and magnetic resonance imaging to produce digital models of animal anatomy well suited to be printed on low-cost 3D printers. In this communication, we report how to apply such technology in comparative anatomy and physiology to aid discovery, description, comprehension and communication, and we seek to inspire fellow researchers in these fields to embrace this emerging technology.

Climate changes can promote disease outbreaks, but their nature and potential impacts in remote areas have received little attention. In a hot spot of biodiversity on the West Antarctic Peninsula, which faces among the fastest changing climates on Earth, we captured specimens of two notothenioid fish species affected by large skin tumors at an incidence never before observed in the Southern Ocean. Molecular and histopathological analyses revealed that X-cell parasitic alveolates, members of a genus we call Notoxcellia, are the etiological agent of these tumors. Parasite-specific molecular probes showed that xenomas remained within the skin but largely outgrew host cells in the dermis. We further observed that tumors induced neovascularization in underlying tissue and detrimentally affected host growth and condition. Although many knowledge gaps persist about X-cell disease, including its mode of transmission and life cycle, these findings reveal potentially active biotic threats to vulnerable Antarctic ecosystems.

A randomized controlled and blinded animal trial was conducted in the axolotl (Ambystoma mexicanum), which has the ability to regenerate from transectional spinal cord injury (SCI). The objective of the present study was to investigate the axolotl's ability to regenerate from a blunt spinal cord trauma in a clinical setting. Axolotls were block-randomized to the intervention (n=6) or sham group (n=6). A laminectomy of two vertebrae at the level caudal to the hind limbs was performed. To induce a blunt SCI, a 25 g rod was released on the exposed spinal cord. Multiple modalities were applied at baseline (pre-surgery), and subsequently every third week for a total of 9 weeks. Gradient echo magnetic resonance imaging (MRI) was applied to assess anatomical regeneration. To support this non-invasive modality, regeneration was assessed by histology, and functional regeneration was investigated using swimming tests and functional neurological examinations. MRI suggested regeneration within 6 to 9 weeks. Histological analysis at 9 weeks confirmed regeneration; however, this regeneration was not complete. By the experimental end, all animals exhibited restored full neurological function. The present study demonstrated that the axolotl is capable of regenerating a contusion SCI; however, the duration of complete regeneration required further investigation.

Bicuspid aortic valve (BAV) is a common congenital heart defect (population incidence, 1-2%)1-3 that frequently presents with ascending aortic aneurysm (AscAA)4. BAV/AscAA shows autosomal dominant inheritance with incomplete penetrance and male predominance. Causative gene mutations (for example, NOTCH1, SMAD6) are known for ≤1% of nonsyndromic BAV cases with and without AscAA5-8, impeding mechanistic insight and development of therapeutic strategies. Here, we report the identification of variants in ROBO4 (which encodes a factor known to contribute to endothelial performance) that segregate with disease in two families. Targeted sequencing of ROBO4 showed enrichment for rare variants in BAV/AscAA probands compared with controls. Targeted silencing of ROBO4 or mutant ROBO4 expression in endothelial cell lines results in impaired barrier function and a synthetic repertoire suggestive of endothelial-to-mesenchymal transition. This is consistent with BAV/AscAA-associated findings in patients and in animal models deficient for ROBO4. These data identify a novel endothelial etiology for this common human disease phenotype.

A new fossil of Ziphiidae from the upper Miocene Gram Formation (ca 9.9-7.2 Ma) is described herein. Computed tomographic scanning of the specimen was performed to visualize the mandibles and to obtain a three-dimensional digital reconstruction. It possesses several characters of the derived ziphiids, such as the dorsoventral thickening of the anterior process of the periotic, the dorsoventral compression of the pars cochlearis and the short unfused symphysis. The specimen cannot be identified beyond the family level, because of the unusual nature of the preserved parts consisting of the mandibles, earbones and postcranial remains. It differs from other ziphiid species from the Gram Formation, Dagonodum mojnum, in its larger size and the more derived morphology of its mandibles and earbones. Its long and thickened stylohyal, combined with its reduced teeth, suggests that this new specimen relied primarily on suction feeding. By contrast, the other ziphiid species from the Gram Formation, D. mojnum, shows adaptations for a more raptorial feeding strategy. Assuming the two species were coeval, their co-occurrence at the same locality with two different feeding strategies, may represent a case of niche separation. They may have hunted different types of prey, thus avoiding direct competition for the same food resource.

Without oxygen, most vertebrates die within minutes as they cannot meet cellular energy demands with anaerobic metabolism. However, fish of the genus Carassius (crucian carp and goldfish) have evolved a specialized metabolic system that allows them to survive prolonged periods without oxygen by producing ethanol as their metabolic end-product. Here we show that this has been made possible by the evolution of a pyruvate decarboxylase, analogous to that in brewer's yeast and the first described in vertebrates, in addition to a specialized alcohol dehydrogenase. Whole-genome duplication events have provided additional gene copies of the pyruvate dehydrogenase multienzyme complex that have evolved into a pyruvate decarboxylase, while other copies retained the essential function of the parent enzymes. We reveal the key molecular substitution in duplicated pyruvate dehydrogenase genes that underpins one of the most extreme hypoxic survival strategies among vertebrates and that is highly deleterious in humans.

Atlantic cod are a commercially important species believed to be threatened by warming seas near their southern, equatorward upper thermal edge of distribution. Limitations to circulatory O2 transport, in particular cardiac output, and the geographic distribution of functionally different haemoglobin (Hb) genotypes have separately been suggested to play a role in setting thermal tolerance in this species. The present study assessed the thermal sensitivity of O2 binding in Atlantic cod red blood cells with different Hb genotypes near their upper thermal distribution limit and modelled its consequences for the arterio-venous O2 saturation difference, Sa-vO2 , another major determinant of circulatory O2 supply rate. The results showed statistically indistinguishable red blood cell O2 binding between the three HbI genotypes in wild-caught Atlantic cod from the Irish Sea (53° N). Red blood cells had an unusually low O2 affinity, with reduced or even reversed thermal sensitivity between pH 7.4 and 7.9, and 5.0 and 20.0°C. This was paired with strongly pH-dependent affinity and cooperativity of red blood cell O2 binding (Bohr and Root effects). Modelling of Sa-vO2  at physiological pH, temperature and O2 partial pressures revealed a substantial capacity for increases in Sa-vO2  to meet rising tissue O2 demands at 5.0 and 12.5°C, but not at 20°C. Furthermore, there was no evidence for an increase of maximal Sa-vO2  with temperature. It is suggested that Atlantic cod at such high temperatures may solely depend on increases in cardiac output and blood O2 capacity, or thermal acclimatisation of metabolic rate, for matching circulatory O2 supply to tissue demand.

The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the α(D)-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the α(A)-globin gene), recurrent losses of α(D)-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa.

The O2 dependence of ouabain-independent K+ transport mechanisms has been studied by unidirectional Rb+ flux analysis in crucian carp red blood cells (RBCs). The following observations suggest that O2 activates K+-Cl- cotransport (KCC) and deactivates Na+-K+-2Cl- cotransport (NKCC) in these cells via separate O2 sensors that differ in their O2 affinity. When O2 tension (PO2) at physiological pH 7.9 was increased from 0 to 1, 4, 21 or 100 kPa, K+ (Rb+) influx was increasingly inhibited, and at 100 kPa amounted to about 30% of the value at 0 kPa. This influx was almost completely Cl- dependent at high and low PO2, as shown by substituting Cl- with nitrate or methanesulphonate. K+ (Rb+) efflux showed a similar PO2 dependence as K+ (Rb+) influx, but was about 4-5 times higher over the whole PO2 range. The combined net free energy of transmembrane ion gradients favoured net efflux of ions for both KCC and NKCC mechanisms. The KCC inhibitor dihydroindenyloxyalkanoic acid (DIOA, 0.1 mM) abolished Cl- -dependent K+ (Rb+) influx at a PO2 of 100 kPa, but was only partially effective at low PO2 (0-1 kPa). At PO2 values between 0 and 4 kPa, K+ (Rb+) influx was further unaffected by variations in pH between 8.4 and 6.9, whereas the flux at 21 and 100 kPa was strongly reduced by pH values below 8.4. At pH 8.4, where K+ (Rb+) influx was maximal at high and low PO2, titration of K+ (Rb+) influx with the NKCC inhibitor bumetanide (1, 10 and 100 microM) revealed a highly bumetanide-sensitive K+ (Rb+) flux pathway at low PO2, and a relative bumetanide-insensitive pathway at high PO2. The bumetanide-sensitive K+ (Rb+) influx pathway was activated by decreasing PO2, with a PO2 for half-maximal activation (P50) not significantly different from the P50 for haemoglobin O2 binding. The bumetanide-insensitive K+ (Rb+) influx pathway was activated by increasing PO2 with a P50 significantly higher than for haemoglobin O2 binding. These results are relevant for the pathologically altered O2 sensitivity of RBC ion transport in certain human haemoglobinopathies.

Videographic material of animals can contain inapparent signals, such as color changes or motion that hold information about physiological functions, such as heart and respiration rate, pulse wave velocity, and vocalization. Eulerian video magnification allows the enhancement of such signals to enable their detection. The purpose of this study is to demonstrate how signals relevant to experimental physiology can be extracted from non-contact videographic material of animals.

Longitudinal animal experiments in the field of regenerative biology often require repeated use of short-term anesthesia (minutes to a few hours). Regain of consciousness limits the level of acceptable invasiveness of procedures, and it makes it difficult to untangle behavioral changes caused by injury to physiological processes involved in the regenerative response. Therefore, a method to keep a regenerative research animal in a comatose state under continuous anesthesia during regenerative experiments often spanning months, would be ethically and experimentally desirable. Here we report on a method using propofol based anesthesia in an isosmotic environment that allows for continuous anesthesia of regenerating axolotls for 60 days with a 75% survival rate, thus spanning the majority of a full regenerative cycle following limb amputation or cryoinjury to the heart. No differences were detected in the axolotl's ability to regenerate amputated limbs and cardiac cryo-injury while anesthetized, however some regenerative failures in the limb were observed in both anesthetized and unanesthetized control groups, most likely caused by prolonged fasting. Sixty days of anesthesia may be approaching a level were kidney function is affected, but the 75% surviving anesthetized animals recovered well after anesthesia and showed a full behavioral recovery within 17 days.

Animal anatomy has traditionally relied on detailed dissections to produce anatomical illustrations, but modern imaging modalities, such as MRI and CT, now represent an enormous resource that allows for fast non-invasive visualizations of animal anatomy in living animals. These modalities also allow for creation of three-dimensional representations that can be of considerable value in the dissemination of anatomical studies. In this methodological review, we present our experiences using MRI, CT and μCT to create advanced representation of animal anatomy, including bones, inner organs and blood vessels in a variety of animals, including fish, amphibians, reptiles, mammals, and spiders. The images have a similar quality to most traditional anatomical drawings and are presented together with interactive movies of the anatomical structures, where the object can be viewed from different angles. Given that clinical scanners found in the majority of larger hospitals are fully suitable for these purposes, we encourage biologists to take advantage of these imaging techniques in creation of three-dimensional graphical representations of internal structures.